Prions are aberrantly folded proteins implicated in the invariably fatal transmissible spongiform encephalopathies (TSEs). TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and several forms of Creutzfeldt-Jakob disease (CJD) in humans. In addition to the TSEs, it has become apparent that other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, contain prion-like behavior in their pathologies. One common feature of these diseases is the aggregation of aberrantly folded proteins and peptides into amyloid fibrils. While the mechanism by which amyloid fibrils are involved in disease is unknown, differences in their molecular structures have been linked with differences in pathologies, a property called the strain phenomenon, which indicates that structure and pathology are interdependent. In order to understand the pathology of these diseases, it is imperative to determine the molecular structures of the implicated amyloids. However, this is a particularly difficult task owing to the heterogeneous and disordered nature of amyloid fibrils. In efforts to develop techniques, in addition to expanding our mechanistic understanding of amyloid formation, the proposed work aims to develop the prion forming domain of the functional fungal prion HET-s as a model system in studying the structure of pathological prions and prion-related amyloids. The HET-s prion forming domain, (218-289), is an ideal model system because infectious fibrils can be formed reproducibly and easily, but the domain still possesses a relatively complex fold comparable to those of pathologically relevant prions. The first aim will be to determine the structure of the infectious form of HET-s(218-289) using joint refinement methods between X-ray fiber diffraction and solid state NMR (ssNMR). The second aim will be to use site- directed mutagenesis to probe the necessity and redundancy of structural elements in correct folding of HET- s(218-289). The third aim will be to characterize infectious and several non-infectious species of HET-s(218- 289), which together can serve as an analog to the strain phenomenon. The methods of characterization will include X-ray fiber diffraction, fluorescence spectroscopy and electron microscopy. These aims will allow for a deeper understanding of the mechanism of prion folding as well as the critical elements necessary for correct folding. Characterization of HET-s(218-289) species, taken with the results of the first two aims, will provide insight into the interplay between environmental conditions and amino acid sequence that determine the final amyloid structure. While these studies cannot replace the need for studies of brain-derived prions, they provide a framework for understanding the mechanisms of prion folding and polymorphism. Preliminary results demonstrate the amenability of HET-s(218-289) to structure determination and biophysical characterization as well as its similarities to and subsequent extensibility in the study of pathological prions. PUBLIC HEALTH RELEVANCE: Prion diseases such as bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD) as well as prion-related diseases such as Alzheimer's and Parkinson's diseases are a continually growing public health concern. The pathologies of these diseases are intricately associated with the molecular structures of aberrantly folded protein aggregates, termed amyloid, that are implicated in these diseases. The proposed studies aim to establish the fungal prion HET-s(218-289) as a model system in efforts to obtain useful structural and mechanistic information that will further advance efforts in the study of disease-associated amyloids.